可定点整合的无筛选标记LacS基因表达载体的构建与鉴定

doi:10.16431/j.cnki.1671-7236.2015.06.004

《中国畜牧兽医》---唯一指定的官方网站 ›› 2015, Vol. 42 ›› Issue (6): 1348-1354.doi: 10.16431/j.cnki.1671-7236.2015.06.004

可定点整合的无筛选标记LacS基因表达载体的构建与鉴定

闫涛, 王瑞瑶, 朱和平, 苏晓田, 王亚娟, 李浩天, 周欢敏

内蒙古农业大学生命科学学院, 呼和浩特 010018

收稿日期:2014-11-06 出版日期:2015-06-20 发布日期:2015-07-23
通讯作者: 周欢敏 E-mail:huanminzhou@263.net
作者简介:闫涛(1989-),男,山东临沂人,硕士,研究方向:发育生物学,E-mail:yta213@163.com
基金资助:
内蒙古家畜繁育生物技术(20091407)

Construction and Identification of Site-specific Integrated and Marker-free LacS Gene Vector

YAN Tao, WANG Rui-yao, ZHU He-ping, SU Xiao-tian, WANG Ya-juan, LI Hao-tian, ZHOU Huan-min

College of Life Sciences, Inner Mongolia Agricultural University, Hohhot 010018, China

Received:2014-11-06 Online:2015-06-20 Published:2015-07-23

摘要/Abstract

摘要： 试验旨在构建一个能够定点整合且无筛选标记基因的半乳糖苷酶基因LacS表达载体.本研究以pEGFP-N1质粒为原始框架,通过PCR及限制性酶切位点在pEGFP-N1质粒的标记基因两端添加两个同向LoxP序列,在多克隆位点上游添加能够定点整合的attB序列,最后再将目的基因BC promoter-LacS-PolyA通过限制性酶切位点插入多克隆位点.每一步都进行PCR、酶切及测序鉴定.PCR产物及酶切片段大小符合预期结果,测序结果也与相应寡核苷酸序列一致,证明各个基因片段正确地连接到载体相应位置.本试验成功构建了可定点整合的无筛选标记半乳糖苷酶基因表达载体pEGFP-N1-LacS.

关键词: 定点整合; 无筛选标记; LacS; pEGFP-N1质粒

Abstract: The objective of this study was to solve the problem of construction a transgenic safety and site-specific integrated and marker-free LacS gene expression vector.Therefore,this experiment using plasmid pEGFP-N1 as the original framework,using PCR and restriction sites to add two with LoxP sequence at both ends of the marker genes in the pEGFP-N1 plasmid,attB sequence was added at the upstream of the multiple cloning sites for site-specific integration,then add the gene BC promoter-LacS-PolyA into the multiple cloning site.By restricted endonucleases digestion,these 3 fragments,Loxp,attB and LacS,were detected in the vector pEGFP-N1-LacS,PCR products and fragment size consistent with the expected results,the results of sequencing and oligonucleotide sequences also corresponding favorable,proved that each gene fragment was correctly connected to the corresponding position of the plasmid.In conclusion,the site-specific integrated and marker-free LacS gene vector was constructed successfully.

Key words: site-specific integrated; marker-free; LacS; pEGFP-N1 vector

中图分类号:

Q782

闫涛, 王瑞瑶, 朱和平, 苏晓田, 王亚娟, 李浩天, 周欢敏. 可定点整合的无筛选标记LacS基因表达载体的构建与鉴定[J]. 《中国畜牧兽医》---唯一指定的官方网站, 2015, 42(6): 1348-1354.

YAN Tao, WANG Rui-yao, ZHU He-ping, SU Xiao-tian, WANG Ya-juan, LI Hao-tian, ZHOU Huan-min. Construction and Identification of Site-specific Integrated and Marker-free LacS Gene Vector[J]. , 2015, 42(6): 1348-1354.

参考文献

[1] Kuroiwa Y,Kasinathan P,Matsushita H,et al.Sequential targeting of the genes encoding immunoglobulinmu and prion pritein in cattle[J].Nature Genetics,2004,36(7):775-780.

[2] Thyagarajan B,Olivares E C,Hollis R P,et al.Site-specific genomic integration in mammalian cells mediated by phage φC31 integrase[J].Journal of Molecular Cell Biology,2001,21(12):3926-3934.

[3] Allen B G,Weeks D L.Transgenic Xenopus laevis embryos can be generated using phi C31 integrase[J].Nature Methods,2005,2(12):975-979.

[4] Karow M,Chavez C L,Farruggio A P,et al.Site-specific recombinase strategy to create induced pluripotent stem cells efficiently with plasmid DNA[J].Stem Cells,2011,29(11):1696-1704.

[5] 朱和平,吴凯峰,苏小虎,等.牛肌肉生长抑制素基因打靶载体的构建与鉴定[J].中国畜牧兽医,2014,41(6):34-37.

[6] 白俊,张东,刘羿羿,等. LacS基因原核表达载体构建及重组蛋白表达[J].上海畜牧兽医通讯,2010,4:4-6.

[7] 曹华明.转基因技术安全性问题和思考[J].天津农业科学,2014,20(3):18-24.

[8] Ma H Y,Ma Q W,Lu Y,et al.Phi C31 integrase induces efficient site-specific recombination in the Capra hircus genome[J].DNA and Cell Biology,2014,33(8):484-491.

[9] Kuhstoss S,Rao R N.Analysis of the integration function of the streptomycete bacteriophage phi C31[J].Journal of Molecular Biology,1991,222(4):897-908.

[10] Rausch H,Lehmann M.Structural analysis of the actinophage phi C31 attachment site[J].Nucleic Acids Research,1991,19(19):5187-5189.

[11] 邹丽婷,宋洪元.噬菌体Phi C31位点重组酶系统在植物转基因中的应用[J].安徽农业科学,2014,42(5):1298-1301.

[12] Park J Y,Choi S U.Optimization of transconjugation and characterization of attB integration site for Streptomyces cinnamoneus producing transglutaminase[J].Biologia,2014,69(8):953-958.

[13] Michel G,Yu Y,Chang T,et al.Site-specifi gene insertion mediated by a Cre-LoxP-carrying lentiviral vector[J].Molecular Therapy,2010,18(10):1814-1821.

[14] 薛健,郭东全,赵桂兰,等.安全选择标记和无选择标记转基因技术研究进展[J].华北农学报,2008,23(增刊):96-102.

[15] 兰翀,任丽娜,吴敏,等.利用Cre/LoxP系统删除转基因山羊体内的选择标记基因[J].生物工程学报,2013,29(12):1847-1854.

可定点整合的无筛选标记LacS基因表达载体的构建与鉴定

Construction and Identification of Site-specific Integrated and Marker-free LacS Gene Vector

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